Xiaohua Lv

Continuously tracing brain-wide long-distance axonal projections in mice at a one-micron voxel resolution.

Revealing neural circuit mechanisms is critical for understanding brain functions. Significant progress in dissecting neural connections has been made using optical imaging with fluorescence labels, especially in dissecting local connections. However, acquiring and tracing brain-wide, long-distance neural circuits at the neurite level remains a substantial challenge. Here, we describe a whole-brain approach to systematically obtaining continuous neuronal pathways in a fluorescent protein transgenic mouse at a one-micron voxel resolution. This goal is achieved by combining a novel resin-embedding method for maintaining fluorescence, an automated fluorescence micro-optical sectioning tomography system for long-term stable imaging, and a digital reconstruction-registration-annotation pipeline for tracing the axonal pathways in the mouse brain. With the unprecedented ability to image a whole mouse brain at a one-micron voxel resolution, the long-distance pathways were traced minutely and without interruption for the first time. With advancing labeling techniques, our method is believed to open an avenue to exploring both local and long-distance neural circuits that are related to brain functions and brain diseases down to the neurite level.

Simultaneous compensation for spatial and temporal dispersion of acousto-optical deflectors for two-dimensional scanning with a single prism

The dispersive nature of the acousto-optical deflector (AOD) presents a challenge to applications of two sequential orthogonal AODs (a two-dimensional AOD) as XY scanners in multiphoton microscopy. Introducing a prism before the two-dimensional (2D) AOD allows both temporal and spatial dispersion to be compensated for simultaneously. A 90 fs laser pulse was broadened to 572 fs without compensation, and 143 fs with compensation. The ratio of long axis to short axis of the exiting laser beam spot was 3.50 without compensation and 1.14 with compensation. The insertion loss was 37%. Two-photon fluorescence microscopy used the compensated 2D AOD scanner to image a fluorescent microsphere, which improves signal intensity -15-fold compared with the uncompensated scanner.

Chemical reactivation of quenched fluorescent protein molecules enables resin-embedded fluorescence microimaging

Resin embedding is a well-established technique to prepare biological specimens for microscopic imaging. However, it is not compatible with modern green-fluorescent protein (GFP) fluorescent-labelling technique because it significantly quenches the fluorescence of GFP and its variants. Previous empirical optimization efforts are good for thin tissue but not successful on macroscopic tissue blocks as the quenching mechanism remains uncertain. Here we show most of the quenched GFP molecules are structurally preserved and not denatured after routine embedding in resin, and can be chemically reactivated to a fluorescent state by alkaline buffer during imaging. We observe up to 98% preservation in yellow-fluorescent protein case, and improve the fluorescence intensity 11.8-fold compared with unprocessed samples. We demonstrate fluorescence microimaging of resin-embedded EGFP/EYFP-labelled tissue block without noticeable loss of labelled structures. This work provides a turning point for the imaging of fluorescent protein-labelled specimens after resin embedding.

Visualization of brain circuits using two-photon fluorescence micro-optical sectioning tomography

Neural circuits are fundamental for brain functions. However, obtaining long range continuous projections of neurons in the entire brain is still challenging. Here a two-photon fluorescence micro-optical sectioning tomography (2p-fMOST) method is developed for high-throughput, high-resolution visualization of the brain circuits. Two-photon imaging technology is used to obtain high resolution, and acoustical optical deflector (AOD), an inertia-free beam scanner is used to realize fast and prolonged stable imaging. The combination of these techniques with imaging and then sectioning method of a plastic-embedded mouse brain facilitated the acquisition of a three-dimensional data set of a fluorescent mouse brain with a resolution adequate to resolve the spines. In addition, the brain circuit tracing ability is showed by several neurons projecting across different brain regions. Besides brain imaging, 2p-fMOST could be used in many studies that requires sub-micro resolution or micro resolution imaging of a large sample.

Construction of multiphoton laser scanning microscope based on dual-axis acousto-optic deflector

The construction of a multiphoton laser scanning microscope based on a dual-axis acousto-optic deflector (AOD) is described. Without mechanical inertia and physical movement of the scanner, this system has a high random-addressing and data-acquisition rate of 10μspixel. By proper compensation of the dispersion of the AODs, the spatial resolution of the system is 0.5μm for lateral direction and 1.5μm for axial direction. Fluorescence images are acquired to demonstrate these imaging performances.

Photostimulation of astrocytes with femtosecond laser pulses

The involvement of astrocytes in brain functions rather than support has been identified and widely concerned. However the lack of an effective stimulation of astrocytes hampers our understanding of their essential roles. Here, we employed 800-nm near infrared (NIR) femtosecond laser to induce Ca2+ wave in astrocytes. It was demonstrated that photostimulation of astrocytes with femtosecond laser pulses is efficient with the advantages of non-contact, non-disruptiveness, reproducibility, and high spatiotemporal precision. Photostimulation of astrocytes would facilitate investigations on information processing in neuronal circuits by providing effective way to excite astrocytes.

NeuroGPS-Tree: automatic reconstruction of large-scale neuronal populations with dense neurites

The reconstruction of neuronal populations, a key step in understanding neural circuits, remains a challenge in the presence of densely packed neurites. Here we achieved automatic reconstruction of neuronal populations by partially mimicking human strategies to separate individual neurons. For populations not resolvable by other methods, we obtained recall and precision rates of approximately 80%. We also demonstrate the reconstruction of 960 neurons within 3 h.

Super-resolution differential interference contrast microscopy by structured illumination.

We propose a structured illumination differential interference contrast (SI-DIC) microscopy, breaking the diffraction resolution limit of differential interference contrast (DIC) microscopy. SI-DIC extends the bandwidth of coherent transfer function of the DIC imaging system, thus the resolution is improved. With 0.8 numerical aperture condenser and objective, the reconstructed SI-DIC image of 53 nm polystyrene beads reveals lateral resolution of approximately 190 nm, doubling that of the conventional DIC image. We also demonstrate biological observations of label-free cells with improved spatial resolution. The SI-DIC microscopy can provide sub-diffraction resolution and high contrast images with marker-free specimens, and has the potential for achieving sub-diffraction resolution quantitative phase imaging.

Violation of the Lagrange invariant in an optical imaging system

The Lagrange invariant provides a basic description of an optical imaging system. Many important conclusions can be drawn from it. We discovered that the Lagrange invariant is violated in a self-interference holography system with particular characteristics. With a proof-of-principle system, we proved this violation both theoretically and experimentally. This finding enables future exciting possibilities in optical imaging.

Analysis of the dispersion compensation of acousto-optic deflectors used for multiphoton imaging

The acousto-optic deflector (AOD) is highly preferred in laser scanning microscopy for its fast scanning ability and random-addressing capability. However, its application in two-photon microscopy is frustrated by the dispersion of the AOD, which results in beam distortion and pulse lengthening. We report the analysis of simultaneous compensation for the angular dispersion and temporal dispersion of the AOD by merely introducing a single dispersive element such as a prism or a grating. Besides serving as a scanner, the AOD is also a part of the compressor pair by integrating the dispersive nature of the AO interaction. This compensation principle is effective for both one-dimensional (1-D) AOD and two-dimensional (2-D) AOD scanning. Switching from a 1-D to a 2-D system requires proper optical alignment with the compensation element, but does not involve any new components. Analytical expressions are given to illustrate the working principle and to help with understanding the design of the system. Fluorescence images of beads and cells are shown to demonstrate the performance of two-photon microscopy when applying this compensated 2-D AOD as scanner.